The production of mice with specific mutations is an important and frequently applied procedure that is used to determine the function of individual genes. For example, genetically altered stem cells are frequently injected into mouse blastocysts by means of cannulas, sharpened needles, or other injection means. The rate at which such injection processes can be performed is severely limited by the time-consuming and intricate production of microinjection needles with which to inject the cells into the blastocysts. In current practice, standard cannulas or needles are converted to injection needles having tips that first must be beveled and then pointed. The fabrication of these improved needles is largely an art requiring experienced technicians and many tedious steps. The inevitable result is that needles are produced that are disturbingly nonuniform. This nonuniformity in turn makes the injection step more demanding, and is a significant impediment to automation. In addition to being time consuming, the lengthy fabrication process also results in many rejects. At times, the defectiveness of the needle becomes apparent only after the whole system, cells and blastocyst are prepared, and the operator attempts an injection under the microscope.
In addition to the above problems caused by having to manually prepare beveled and tipped needles, the manner in which the blastocyst injection step is carried out presents additional difficulties. Typically, the injection is done on the stage of an inverted microscope. There are usually one or more micromanipulators rigidly attached to the microscope. These are used to manipulate the biological materials and perform other operations. The micromanipulators typically have a set of three motors used for coarse positioning, and a hydraulic actuator operated from a joystick that is used for fine positioning. The micromanipulators have been developed to the point where they are capable of positioning the tip of the needle at the blastocyst wall very well. It is also known to employ a computer to return the injection needle to a previous microposition after it has been withdrawn.
The injection step is usually carried out manually with a micromanipulator. The usual practice is for the operator to position the injection needle at the wall of the blastocyst, pull the needle back out of sight from the viewing field of the microscope in order to allow enough distance to accelerate the needle, and then slap the handle of the micromanipulator forward to jam the needle tip into the blastocyst. If the hand of the operator does not move along a sufficiently straight line, the blastocyst may be missed, knocked away, or torn and destroyed by the sharp needles. These attempts to use micromanipulators for the injection step have thus generally been unsatisfactory. No micromanipulators are known that are capable of carrying out the precise thrusting motion over the requisite microscopic distance needed to achieve proper cell injection.
In the above-described injection methods, a further complicating aspect is that of stopping the advance of the injection needle a predetermined distance into the blastocyst. A fully satisfactory solution to this problem has not been achieved by the known methods either.